Turbot (Scophthalmus maximus L.) occludin gene performs the anti-inflammatory function in the intestine via the regulations by miR-140-3p in the NF-κB pathway

Abstract

Occludin (OCLD), as a vital member of TJ superfamily, plays important regulatory roles in the processes of proliferation, repair and survival of epithelial cells following various stresses. In teleost, OCLD gene was demonstrated to regulate the permeability near epithelial cells to be involved in the mucosal immune response. Even though the immune functions of OCLD genes were investigated in mammals and a part of fish species, no relevant studies have been done on the potential mechanism of the anti-inflammatory regulations of OCLD in the turbot mucosal immunity during bacterial infection, moreover, with the regulation of miRNAs to turbot OCLD. In this study, the full-length sequence of OCLD nucleotide was cloned and confirmed by PCR amplification reaction. Following bacterial infections, the mRNA expressions of OCLD and genes related to OCLD were detected in the turbot intestinal tissues and cells following bacterial infections. Afterward, knockdown of OCLD in turbot intestine cells (SMI cells) was used to confirm the key roles of OCLD in the mucosal immune system, as well as cell apoptosis. The promotions of cell proliferation and migration of turbot OCLD were confirmed in the SMI cells for wound healing. The target regulationship between miR-140-3P and turbot OCLD was predicted and verified by luciferase reporter assay. Finally, the anti-inflammatory functions of turbot OCLD were investigated in the OCLD knockdown SMI cells (in vitro), which was further verified in zebrafish model with DSS-induced colitis (in vivo) through recombinant turbot OCLD protein. Therefore, we hypothesized that turbot OCLD gene could be regulated by miR-140-3p to attenuate the apoptosis of SMI cells, thereby suppressing the inflammation in the intestine via the regulations of the NF-κB signaling pathway. This study contributes to the fundamental and advanced understanding of the immune regulatory functions of turbot OCLD in the intestinal inflammation, which will also provide the new sights for the studies on the prevention and treatment of fish diseases during bacterial invasion on the mucosal system.